CN113754751B - 一种新型促睡眠乳源活性肽ccl-2s及其制备方法和应用 - Google Patents
一种新型促睡眠乳源活性肽ccl-2s及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种新型促睡眠乳源活性肽CCL‑2S,其氨基酸序列为FALPQYLK。本发明以乳源酪蛋白酶解物为原料,采用脱盐等基础手段除杂后,利用体外膜片钳技术从牛奶中分离鉴定出一种具有抑制神经细胞动作电位发放频率、能促进睡眠的新型促睡眠乳源活性肽CCL‑2S。同时,本发明提供的新型促睡眠乳源活性肽安全无毒,可应用于制备促睡眠功能性食品等产品,可充分利用乳产品资源,为乳源多肽的高值化利用提供了新途径。
Description
技术领域
本发明属于乳源有效成分提取技术领域,具体涉及一种新型促睡眠乳源活性肽CCL-2S及其制备方法和应用。
背景技术
睡眠约占人一生中三分之一的时间,良好的睡眠是身心健康的前提保障。随着现代社会的发展,人们的生活节奏普遍加快,各方面压力等造成失眠的现象越来越多。睡眠不足会直接导致人们思维迟缓、头脑紧张、注意力不集中、行动迟缓、厌烦、忧虑、倦怠,对工作缺乏热情,使工作主动性与工作意愿下降,从而降低工作效率。长期以往,睡眠不足的人会感到特别的疲惫辛苦,情绪不稳定,暴躁、易怒,缺乏自制力,造成心理方面严重的问题。此外,失眠还会导致抵抗力下降,患病机率大。
目前,临床上常用化学合成类镇静催眠药,例如以苯二氮䓬类为主的镇静促睡眠药,可治疗失眠及其他疾病引起的睡眠障碍。但长期服用这类药物会出现多种副作用,患者对该类药物易形成依赖。因此,寻找一种药效显著、副作用小、安全的改善睡眠药物是医药卫生领域的一项重要任务。
牛奶具有良好的促睡眠效果,牛乳中含有多种生物活性多肽,但天然牛乳中活性多肽的含量较少,无法满足亚健康人们的需要。牛乳酪蛋白,是许多具有潜在生物活性物质的前体,通过蛋白酶的酶解作用,可以得到具有生物学功能的乳源生物活性肽。这些小肽一般由2-20个氨基酸残基组成,能够对机体的消化系统、免疫系统、心血管系统等多方面产生积极的影响,具有调节神经系统、抗菌、抗氧化、抗高血压、免疫调节等生理活性。
中国发明专利CN202010387572.5公开了一种乳源活性肽CCL-3S在制备促睡眠产品中的应用,利用体外膜片钳技术从牛奶中分离鉴定出一种具有抑制神经细胞动作电位发放频率、能促进睡眠的乳源活性肽CCL-3S,该乳源活性肽氨基酸序列为HQGLPQEVLNENLLR,分子量为1759.998Da,作用于细胞后,Vm的变化率为12.56± 3.67%,与空白组比较,具有显著性抑制作用(p<0 .05),能显著抑制神经细胞动作电位发放频率。
发明内容
本发明的目的在于提供一种新型促睡眠乳源活性肽CCL-2S,能显著抑制神经细胞动作电位发放频率,有来源安全、促睡眠效果好、活性稳定等优点。
本发明通过以下技术方案实现:
一种新型促睡眠乳源活性肽CCL-2S,包括如SEQ ID NO:1所示的氨基酸序列,Phe-Ala-Leu-Pro-Gln-Tyr-Leu-Lys(FALPQYLK),分子量为979.17,可用于制备促睡眠产品,即功能性食品或者药品。
本发明新型促睡眠乳源活性肽CCL-2S的制备方法,包括以下步骤:
(1)原料预处理:浓缩乳源酪蛋白酶解物原料,得到多肽饱和水样,采用真空喷雾干燥,得到多肽粉末;
(2)C18制备柱一次分离
将所得多肽粉末用超纯水配制成200mg/mL的溶液,过0.22 μm滤膜后,借助制备型高效液相色谱对促睡眠肽进行初步分离;
根据图谱中出峰时间及峰形相似度,分为6段进行接样,将这6段组分分别收集,尽快进行浓缩(60℃旋蒸)及真空冻干,随后测量其体外促睡眠活性;
色谱条件:色谱柱为自装玻璃柱(20 mm×450 mm,C18填料(10μm,300 Å,MachereyNagel,France));流动相:A 泵为超纯水(含0.1% TFA),B 泵为乙腈(含0.1% TFA);流速:10mL/min;进样量:5 mL;检测波长:214 nm;洗脱条件:10%-20%(0.01-30 min),20%-35%(30-90 min),35%-52.5%(90-110 min),52.5%-90%(110-125 min);
(3)C18制备柱二次分离
将具有最好的促睡眠活性的一段组分用超纯水配制成200mg/mL的溶液,过0.22 μm滤膜后,借助制备型高效液相色谱对其进行初步分离;
根据图谱中出峰时间及峰形相似度,分为3段进行接样,将这3段组分分别收集,尽快进行浓缩(60℃旋蒸)及真空冻干,随后测量其体外促睡眠活性;
色谱条件:色谱柱为Welch C18 柱子(4.6 mm×250 mm,5 μm,Ultimate);流动相:A泵为超纯水(含0.1% TFA),B泵为乙腈(含0.1% TFA);进样量:20 µL;流速:1 mL/min;检测波长:214 nm;洗脱条件:10%-20%(0.01-10 min),20%-30%(10-40 min),30%-90%(40-48min);
(4)HCLP的第三次分离
将具有最好的促睡眠活性的一段组分用超纯水配制成200mg/mL的溶液,过0.22 μm滤膜后,借助制备型高效液相色谱对其进行初步分离;
根据图谱中出峰时间及峰形相似度,分为4段进行接样,将这4段组分分别收集,尽快进行浓缩(60℃旋蒸)及真空冻干,随后测量其体外促睡眠活性,具有最好的促睡眠活性的一段即为本发明新型促睡眠乳源活性肽CCL-2S;
色谱条件:色谱柱为Welch C18 柱子(4.6 mm×250 mm,5 μm,Ultimate);流动相:A泵为超纯水(含0.1% TFA),B泵为乙腈(含0.1% TFA);进样量:20 µL;流速:1 mL/min;检测波长:214 nm;洗脱条件:10%-20%(0.01-10 min),20%-30%(10-40 min),30%-90%(40-48min)。
本发明具有以下积极有益效果:
本发明新型促睡眠乳源活性肽安全无毒,能更加显著地抑制神经细胞动作电位发放频率,Vm变化率达21.47±2.66,AP的抑制率达29.41±8.79,具有优异的促睡眠活性,与空白组比较,具有显著性抑制作用(p<0.01);可应用于制备促睡眠功能性食品等,并可充分利用乳产品资源,为乳源多肽的高值化利用提供了新途径。
附图说明
图1为制备液相一次分离分段接样图谱;
图2为制备液相一次分离样品AP抑制率;
图3为制备液相一次分离样品Vm的变化率;
图4为制备液相二次分离分段接样图谱;
图5为制备液相二次分离样品AP抑制率;
图6为制备液相二次分离样品Vm的变化率;
图7为P3中段分析液相图谱;
图8 生理条件下诱发的VLPO的AP;
图9 CCL-2S对VLPO神经元诱发的AP抑制;
图10 CCL-2S氨基酸测序仪图谱。
具体实施方式
为了更好的了解本发明,下面通过具体的实施例来说明本发明的技术方案。以下实施例中所用药品和试剂均为市购,所用乳源酪蛋白酶解物为牛乳酪蛋白采用蛋白酶酶解后的牛乳多肽溶液,具体制备方法如下:将酪蛋白与蒸馏水以 1:10 (w/v) 的比例混合,再将0.5%的胰蛋白酶加入酪蛋白液中水解5小时,将pH 值调至4-5,离心取上清,然后,将酶解产物(上清液)高温灭活,随后离心收集上清液,冻干,-20℃保存备用。
本发明新型促睡眠乳源活性肽,由以下方法筛选制备:
(1)原料预处理:将乳源酪蛋白酶解物原料经旋转蒸发浓缩,得到多肽饱和水样,采用真空喷雾干燥,得到多肽粉末。
(2)C18制备柱一次分离
将多肽粉末(含有大量促睡眠肽)用超纯水配制成200mg/mL的溶液,使用注射器抽取并过0.22 μm滤膜后,借助制备型高效液相色谱(LC-8A, Shimadzu)对促睡眠肽进行初步分离。
色谱条件:色谱柱为自装玻璃柱(20 mm×450 mm,C18填料(10μm,300 Å,MachereyNagel,France));流动相:A 泵为超纯水(含0.1% TFA),B 泵为乙腈(含0.1% TFA);流速:10mL/min;进样量:5 mL;检测波长:214 nm,;洗脱条件:10%-20%(0.01-30 min),20%-35%(30-90 min),35%-52.5%(90-110 min),52.5%-90%(110-125 min)。
多肽粉末在制备型高效液相色谱中分离检测的图谱如图1所示,多肽常用检测波长214nm下,根据出峰时间及峰形相似度,分为6段进行接样,命名为P1、P2、P3、P4、P5、P6。将这6段组分分别收集,尽快进行浓缩(60℃旋蒸)及真空冻干,减少多肽降解。随后测量其体外促睡眠活性,对离体大鼠VLPO神经元Vm及去极化电流诱发AP的影响,结果如图2和图3所示。因P5、P6样品不溶于脑脊液,故分析P1-P4组分发现P3组分抑制VLPO神经元的兴奋性效果最好,其他组分抑制效果并不明显,故选择P3组分进行下一步的分离和分析。
体外促睡眠活性的测定运用膜片钳技术。首先制作下丘脑切片,取出生5周左右的大鼠,体重约100g-150g,急性分离其下丘脑,将下丘脑切片为400μm,接着制备微电极,将切片迅速放入充满95%O2和5%的人工脑脊液中,在33℃中孵育30min,拿出孵育槽,在室温下放置20min。采用外径为1.4mm的有芯硬质玻璃毛坯,抛光后尖端拉制为直径约2μm,充满电极内液。然后将微电极与下丘脑切片的VLPO神经元进行封接,用5× 的物镜观察下丘脑的结构,找到腹外侧视前区,然后用40× 浸水镜观察单个腹外侧视前区神经元的形态并利用电极进行封接,当电极尖端与细胞间形成大于1GΩ封接后成功后,在封接窗口观察到细胞出现电容充放电后,即形成全细胞模式。首先记录一段自发放电作为实验对照,通常2-3min,膜电位50mV,稳定后给药。然后灌流含牛乳多肽的灌流液,以观察神经元放电形式的变化,通过评价Vm的变化率与去极化电流诱发AP的个数评估VLPO神经元兴奋性抑制率的影响,从而分析多肽的体外促睡眠活性。
(3)C18制备柱二次分离
根据以上研究发现P3具有较好的促睡眠活性,将P3组分用超纯水配制成200mg/mL的溶液,使用注射器抽取并过0.22 μm滤膜后,借助制备型高效液相色谱(LC-8A,Shimadzu)对其进行初步分离。P3组分在制备型高效液相色谱中分离检测的图谱如图4所示,多肽常用检测波长214nm下,根据出峰时间及峰形,将P3组分分为前中后三段,浓缩冻干,继续评价体外促睡眠活性。发现经过进一步分离P3组分中段具有更好的促睡眠活性,见图5和图6。
色谱条件:色谱柱为Welch C18 柱子(4.6 mm×250 mm,5 μm,Ultimate);流动相:A泵为超纯水(含0.1% TFA),B泵为乙腈(含0.1% TFA);进样量:20 µL;流速:1 mL/min;检测波长:214 nm;洗脱条件:10%-20%(0.01-10 min),20%-30%(10-40 min),30%-90%(40-48min)。
(4)HCLP的第三次分离
根据以上研究发现P3中段具有较好的促睡眠活性,用以上同样的方法将P3中段分为4个单峰,浓缩冻干,进行体外促睡眠活性探索,见图7-9和表1,发现单体2(CCL-2S)的Vm变化率为21.47±2.66,AP的抑制率为29.41±8.79,具有良好的促睡眠活性。
色谱条件:色谱柱为Welch C18 柱子(4.6 mm×250 mm,5 μm,Ultimate);流动相:A泵为超纯水(含0.1% TFA),B泵为乙腈(含0.1% TFA);进样量:20 µL;流速:1 mL/min;检测波长:214 nm,280 nm;洗脱条件:10%-20%(0.01-10 min),20%-30%(10-40 min),30%-90%(40-48 min)。
表1 单体2对VLPO神经元兴奋性的影响(n=11)
注:与正常对照组比较,*p<0.05,**p<0.01。
(5)促睡眠乳源活性肽的一级结构鉴定
利用氨基酸测序仪(PPSQ)测定CCL-2S多肽段N端绝对序列,见图10,通过比对Uniprot数据库中牛乳酪蛋白蛋白序列,确定多肽的序列为Phe-Ala-Leu-Pro-Gln-Tyr-Leu-Lys(FALPQYLK),来源于牛乳酪蛋白中的Alpha-S2-酪蛋白,这是首次从食物源天然产物中分离出此序列促睡眠肽。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 华南农业大学
<120> 一种新型促睡眠乳源活性肽CCL-2S及其制备方法和应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8
<212> PRT
<213> 多肽(polypeptide)
<400> 1
Phe Ala Leu Pro Gln Tyr Leu Lys
1 5
Claims (1)
1. 一种新型促睡眠乳源活性肽CCL-2S在制备促睡眠产品中的应用,所述新型促睡眠乳源活性肽CCL-2S的氨基酸序列如SEQ ID NO:1所示;
所述促睡眠为抑制神经细胞动作电位发放频率。
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